Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at ?80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at ?20 °C for 1 year (FD-20). In a separate experiment, EDTA plasma samples were collected onto ice, processed ice cold and maintained on ice ± protease inhibitors versus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography and tandem mass spectrometry (LC–ESI–MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST algorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at room temperature. Random sampling by LC–ESI–MS/MS showed that peptides from C4B were undetectable on ice, but peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room temperature. The frequency and intensity of precursors within ± 3 m/z of the C4B peptide NGFKSHALQLNNR was confirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target sequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature chain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the mature C4B protein, to reveal the thioester reaction site, consistent with LC–ESI–MS/MS and Western blot. Random sampling showed that proteolytic peptides from complement component C4B were rarely observed with long term storage at ? 80 °C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at ? 20 °C (FD-20 °C) or freeze drying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at least 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to samples on ice for up to 72 h or stored at ? 80 °C, LN2, FDRT or FD-20 °C for up to a year.     

Detection of Urinary Excreted Fungal Galactomannan-like Antigens for Diagnosis of Invasive Aspergillosis

Mortality associated with invasive aspergillosis (IA) remains high, partly because of delayed diagnosis. Detection of microbial exoantigens, released in serum and other body fluids during infection, may help timely diagnosis. In course of IA, Aspergillus galactomannan (GM), a well established polysaccharide biomarker, is released in body fluids including urine. Urine is an abundant, safely collected specimen, well-suited for point-of-care (POC) testing, which could play an increasing role in screening for early disease. Our main objective was to demonstrate GM antigenuria as a clinically relevant biological phenomenon in IA and establish proof-of-concept that it could be translated to POC diagnosis. Utilizing a novel IgM monoclonal antibody (MAb476) that recognizes GM-like antigens from Aspergillus and other molds, we demonstrated antigenuria in an experimental animal IA model (guinea pig), as well as in human patients. In addition, we investigated the chemical nature of the urinary excreted antigen in human samples, characterized antigen detection in urine by immunoassays, described a putative assay inhibitor in urine, and indicated means of alleviation of the inhibition. We also designed and used a lateral flow immunochromatographic assay to detect urinary excreted antigen in a limited number of IA patient urine samples. In this study, we establish that POC diagnosis of IA based on urinary GM detection is feasible. Prospective studies will be necessary to establish the performance characteristics of an optimized device and define its optimal clinical use.     

Genome-Wide Analysis of the Fusarium oxysporum mimp Family of MITEs and Mobilization of Both Native and De Novo Created mimps

We have performed a genome-wide analysis of the mimp family of miniature inverted-repeat transposable elements, taking advantage of the recent release of the F. oxysporum genome sequence. Using different approaches, we detected 103 mimp elements, corresponding to 75 nonredundant copies, half of which are located on a single small chromosome. Phylogenetic analysis identified at least six subfamilies, all remarkably homogeneous in size and sequence. Based on high sequence identity in the terminal inverted repeats (TIRs), mimp elements were connected to different impala members. To gain insights into the mechanisms at the origin and amplification of mimps, we studied the potential of impala to cross-mobilize different mimps, native but also created de novo by inserting a short DNA segment between two TIRs. Our results show that TIR sequences are the main requirement for mobilization but that additional parameters in the internal region are likely to influence transposition efficiency. Finally, we show that integration site preference of native versus newly transposed mimps greatly varies in the host genomes used in this study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequences of novel mimp3 and mimp4 elements are available under GenBank accession numbers EU833100 and EU833101, respectively. Coordinates of mimp5, mimp6 and of non-classified mimp copies are indicated in Supplementary Table 1.     

FRET hybridization probes for the rapid detection of disease resistance alleles in plants: detection of corky root resistance in lettuce

We describe the development of a non-electrophoresis PCR-based assay for allele discrimination at a disease resistance locus. The assay is based on the emission of light by fluorescence resonance energy transfer (FRET) upon annealing of two hybridization probes. The analysis of melting curve profiles of the probes and templates allowed the detection of single nucleotide polymorphisms. The assay was applied to the detection of alleles at the cor locus in lettuce (Lactuca sativa) that confers recessive resistance to corky root disease. Probes and primers for the assay were designed after the characterization of a single nucleotide polymorphism between alleles of PCR products amplified using a linked marker. That polymorphism was validated in a collection of lettuce varieties representing different genetic backgrounds. The FRET hybridization probes approach provided fast and accurate genotyping of breeding material directly in a one-tube reaction. The absence of electrophoresis makes this approach suitable for applications that require automation and high-throughput genotyping analyses such as marker-assisted selection programs.     

Case Series: Report of the First Two Human Indigenous Cases of <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> Infection in Eastern Canada

We report the two first cases of human C. gattii meningoencephalitis acquired on the Canadian east coast, from the province of Quebec. Unlike C. neoformans, C. gattii is not known to have an established ecological niche on the North American east coast. C. gattii has recently been responsible for major outbreaks in British Columbia, Canada, and in the American pacific northwest. However, no human cases acquired in other Canadian provinces have been reported to our knowledge. The source of acquisition remains unclear for both patients but since neither had traveled outside of the province of Quebec, we discuss the possibilities of environmental and animal-associated acquisition, as well as the possible established endemicity in new areas. These cases add to the growing reported human and animal cases in areas previously not thought to be endemic for C. gattii.